Dioxygenases catalyze the incorporation of both atoms of molecular oxygen into a substrate. Microbial dioxygenases are absolutely critical for the detoxification and degradation of aromatic found in the environment because they catalyze the key metabolic step - opening of the aromatic ring. In mammals, dioxygenases are involved in the synthesis of tryptophan and of leukotrienes and in the metabolism of the aromatic amino acids. The focus of this work will be on protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas aeruginosa. This is the only dioxygenase for which the structure has been determined (Ohlendorf et al, 1988) and its refinement will be pursued. In addition, a number of inhibitors and substrates previously analyzed spectroscopically will be soaked into crystals to provide detailed structural data for the mechanistically relevant complexes. The gene for 3,4-PCD will be cloned and expressed to explore the function of residues in the active site as well as to restore activity of the vestigial active site. The structural analysis suggests that this site was functional in the ancestral dioxygenase. The immediate goal of this project is to gain understanding of how dioxygenases function through detailed structural analysis and site-directed mutagenesis combined with the spectroscopic evidence amassed by members of this research team. The longer term goal is to better understand the evolution of catalysis through the analysis and modification of functional and vestigial active sites found in the dioxygenases.